The effects of iron on the cell cycle of HeLa cells

Previous studies have shown that when nonsynchronous HeLa cells in the logarithmic growth phase are incubated in an iron rich medium, the rate of cell multiplication decreases after 24 hours of iron incubation. The present experiments were designed to determine the phase of the cell cycle in which iron exerts its negative effect on cell growth. Randomized populations of HeLa cells growing on slides were incubated with ferrous ammonium sulfate at final concentrations of 0.0035 and 0.00175 g/ml. Cell cycle analysis was accomplished using light microscopic autoradiography by counting mitoses labeled with radioactive thymidine (³H thy) in cells incubated in iron from 0 to 36 hours. In a second group of experiments, control and iron incubated cells were labeled with ³H thy, and the percent labeled nuclei was recorded hourly after 0 to 7 hours. A third group of cells was incubated with iron from 0 to 7 hours following pulse labeling with ³H thy. These were fixed at hourly intervals after a second pulse label, and the percent labeled nuclei was counted. By selective detachment of metaphase cells, a synchronous population of HeLa cells in the G₁ period was obtained. These cells were also incubated with ferrous ammonium sulfate at the above indicated concentrations and fixed after pulse labeling with ³H thy. The results suggest that iron affects the cell cycle of HeLa cells by lengthening it by 4 to 5 hours. The negative effect of iron seems to be a block in the late G1 period, about 2 to 4 hours before the initiation of DNA synthesis. The possibility of a second block in the G₂ phase cannot be ruled out.